IDENTIFYING AMINO-ACID-RESIDUES THAT INFLUENCE PLASMA-CLEARANCE OF MURINE IGG1 FRAGMENTS BY SITE-DIRECTED MUTAGENESIS

Site-directed mutagenesis has been used to change amino acid residues of a recombinant Fc-hinge fragment derived from the murine immunoglobu lin (Ig)G1 molecule, and the effects of these mutations on the pharmac okinetics of the Fc-hinge fragment have been determined. Specifically, Ile-253, His-310 and Gln-311 of the CH2. domain and His-433 and Asn-4 34 of the CH3 domain have been changed. In the three dimensional struc ture of an antibody, these amino acids art in close proximity to each other at the CH2-CH3 domain interface. The mutated Fc-hinge fragments have been purified from recombinant Escherichia coli cells and their p harmacokinetic parameters determined in mice and compared with those o f the wild-type Fc-hinge fragment. The results show that the site of t he IgG1 molecule that controls the catabolic rate (the 'catabolic site ') is located at the CH2-CH3 domain interface and overlaps with the St aphylococcal protein A binding site.